ABSTRACT
WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.
Subject(s)
Oxidoreductases/physiology , Serine/chemistry , Staurosporine/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Protein p53/metabolism , Tyrosine/chemistry , Active Transport, Cell Nucleus , Animals , Anisomycin/pharmacology , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Cytosol/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Etoposide/pharmacology , Fibroblasts/metabolism , Genes, Dominant , Humans , Hypoxia , Imidazoles/metabolism , Immunoprecipitation , Luminescent Proteins/pharmacology , Mice , Microscopy, Fluorescence , Models, Biological , Phosphorylation , Piperazines/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Suppressor Proteins , Two-Hybrid System Techniques , U937 Cells , Ultraviolet Rays , WW Domain-Containing OxidoreductaseABSTRACT
By cDNA library screening, here we isolated an unusual gene transcript encoding a 31-amino-acid zinc finger-like peptide that regulates apoptosis (named Zfra). Northern blotting and RT/PCR showed the transcript is abundant in spleen but absent in several prostate and breast cancer cells. When stably expressed in L929 fibroblasts, Zfra conferred resistance to the cytotoxic effects of TNF and FasL. In contrast, transiently expressed Zfra could enhance or inhibit the cytotoxicity of overexpressed death domain proteins TRADD, FADD, and RIP of the TNF signaling pathway. By GST pull-down assay and co-immunoprecipitation, TNF and UV light were shown to induce Zfra to rapidly self-associate and bind JNK1. While JNK1 is a downstream effector of the TNF signaling, Zfra regulation of the TNF cytotoxic function is likely due to its interaction, in part, with JNK1.